The constitutive activation of the Wnt signaling pathway plays a major role in the resistance of cancer cells to current therapeutic agents. The interaction of beta- catenin and BCL-9 is critical for the constitutive activation of the Wnt signaling pathway. Recent determination of a high-resolution crystal structure of beta-catenin and BCL-9 suggests that unlike most other protein-protein interactions, the beta-catenin and BCL-9 interaction is mediated by a well-defined binding pocket in beta-catenin and several key hydrophobic residues that are clustered together. Thus, the beta-catenin and BCL-9 interaction may be targeted by non-peptidic, small-molecules. To this end, we have carried out extensive mutational studies on BCL-9 and have defined the key binding residues. Furthermore, we have developed robust fluorescence-polarization and FRET assays for this interaction. In this R21 grant, we propose to carry out high throughput screening for the discovery of small-molecule antagonists of the beta-catenin-BCL-9 interaction. Identified "hits" will be further investigated for their specificity, their mechanism of action and for their cellular activity and specificity. It is expected that successfully carried out;this project will lead to the development of a number of assays for high throughput screening for the discovery of bona fide small-molecule antagonists of the beta-catenin-BCL-9 interactions. PUBLIC HEALTH RELEVANCE: Cancer is the second leading cause of death in the United State of America. More effective treatments are urgently needed to improve the outcome of millions of cancer patients. This research project aims at development of high throughput screening assays for the beta-catenin-BCL-9 protein-protein interaction and discovery of non-peptide, small-molecule inhibitors of this interaction. These small-molecule inhibitors will serve as important lead compounds for further optimization, which can lead to the development of a new class of small-molecule anti-cancer drugs for the treatment of human cancer, including but not limited to breast cancer and prostate cancer.